Design, synthesis, and in silico studies of quinoline-based-benzo[d]imidazole bearing different acetamide derivatives as potent α-glucosidase inhibitors

In this study, 18 novel quinoline-based-benzo[d]imidazole derivatives were synthesized and screened for their α-glucosidase inhibitory potential. All compounds in the series except 9q showed a significant α-glucosidase inhibition with IC50 values in the range of 3.2 ± 0.3–185.0 ± 0.3 µM, as compared to the standard drug acarbose (IC50 = 750.0 ± 5.0 µM). A kinetic study indicated that compound 9d as the most potent derivative against α-glucosidase was a competitive type inhibitor. Furthermore, the molecular docking study revealed the effective binding interactions of 9d with the active site of the α-glucosidase enzyme. The results indicate that the designed compounds have the potential to be further studied as new anti-diabetic agents.

www.nature.com/scientificreports/ the reaction of O-phenylenediamines (4) and 3-formyl-2-mercaptoquinoline (3) in the presence of sodium metabisulfite in DMF at 150 °C for 2 h afforded the target compound 5 33 . Synthesis of desired compounds 8a-r was performed through the reaction of aniline derivatives (6a-r) with chloroacethylchloride (7) in DMF 34 . Finally, the reaction of compounds 8a-r and compound 5 in acetone in presence of K 2 CO 3 led to the formation of products 9a-r 35 . The structure of all compounds was confirmed using NMR and IR spectroscopy as well as elemental analysis.

Structure-activity relationship (SAR) exploration.
The results of the α-glucosidase inhibitory assay are displayed in Table 1. In general, all compounds showed significant α-glucosidase inhibition with IC 50 values in the range of 3.2 ± 0.3 to 185.0 ± 0.3 µM in comparison to acarbose with an IC 50 value of 750.0 ± 5.0 µM. The exception come back to 9q which showed IC 50 > 750. As can be seen in Table 1, benzimidazole-thioquinoline structure bearing phenylacetamide exhibited good inhibitory activities against α-glucosidase (9a, IC 50 = 30.2 ± 0.4 µM). The incorporation of a fluorine atom at the ortho position of phenylacetamide (9b) resulted in an around the twofold loss of potency compared to 9a. Furthermore, changing the position from ortho to para in compound 9c (IC 50 = 13.5 ± 0.6 µM) resulted in the second potent derivative in the halogen-substituted set.
Overall, the mono-electron withdrawing group (EWG) at para position had a destructive effect against α-glucosidase while ortho and meta position seems more favorable. The exception in this trend came back to 9c bearing 4-fluorine. This could be due to the smaller size and better electronegativity compared to the rest of halogen derivatives.
The evaluations on 9j-m as the mono electron-donating-substituted group (EDG) showed overall improvement in the potency so that 9l (R = 4-methoxyphenyl) with an IC 50 of 5.7 ± 0.3 μM was categorized as the top potent inhibitor in this group and second top potent entry among all derivatives followed by 9k (R = 4-methyl phenyl) and 9j (R = 2-methyl phenyl). Next, the assessment of compounds 9n and 9o were performed and,  Ring substitution assessments were also performed in which phenyl (9a) was replaced with naphthyl (9p). An improvement in the activity showed that a bulk structure is more favorable.
Next, the investigation of SAR indicated that nitro (9i, IC 50 = 19.7 ± 0.2 µM) and methoxy (9l, IC 50 = 5.7 ± 0.3 µM) moieties were optimal substituents at the para position of phenylacetamide which improved the α-glucosidase inhibition. These results suggested that such substitution may probably enhance the ligand-protein interaction with the α-glucosidase active site.
9q and 9r were also synthesized to evaluate the role of elongation of the linker between aryl substitutions and thioacteamide moiety. Compound 9q with benzyl substitution exhibited dramatically reduction in the α-glucosidase inhibition compared to 9a which exhibited the destructive effect of elongation of the linker in the unsubstituted derivatives. Also, there was a similar trend in the potency in 9r bearing 4-fluorobenzyl compared to 9c (R = 4-Fluorophenyl).
The summary of the SARs to improve α-glucosidase inhibitory activity was depicted in Fig. 3. Overall, it can be understood that the most potent derivative (9d) exhibited better inhibitory activity against a-glucosidase compared to lead compounds including A to G reported in Fig. 1 concerning their positive control.
Enzyme kinetic studies. To gain insight into the mechanism of action of 9d as the most potent α-glucosidase inhibitor, kinetic measurements were performed. According to Fig. 4a, the Lineweaver-Burk plot showed that the K m gradually increased and V max remained unchanged with increasing inhibitor concentration indicating a competitive inhibition. The results show 9d bonded to the active site on the enzyme and compete with the substrate for binding to the active site. Furthermore, the plot of the K m versus different concentrations of inhibitor gave an estimate of the inhibition constant, K i of 3.2 µM (Fig. 4b).
Docking analyses. To identify the accuracy and validation of docking procedures, the self-docking of acarbose (as a crystallographic ligand) was performed through induced fit docking of Schrödinger software. Alignment of the best pose of acarbose in the active site of α-glucosidase and crystallographic ligand recorded an RMSD value of 1.73 Å (RMSD should be less than 2 Å) which confirms the accuracy of docking. Next, the same Table 1. α-Glucosidase inhibitory activity of compounds 9a-r. a Data represented in terms of mean ± SD.   www.nature.com/scientificreports/ docking procedure was repeated with all derivatives and their binding to α-glucosidase was analyzed. Results are summarized in Table 2.
The docking results between α-glucosidase and compound 9d was shown in Fig. 5. Compound 9d was well inserted into the active site and recorded a Glide score of − 6.92. Compound 9d established critical hydrogen bond interaction with Trp481 and benzimidazole. Also, benzimidazole participated in pi-cation interaction with Arg600. On the other side of the molecule, 3-chlorophenylacetamide established H-bound interaction with Asp616 and halogen-bound interaction with Leu677. Notably, in most derivatives, the designed scaffold participated in the critical interactions within the active site of the enzyme and showed similar kinds of interactions to the native ligand.

Conclusion
In this study, a series of novel quinoline-based-benzo[d]imidazole bearing different acetamide derivatives were designed, synthesized and their inhibitory activity against α-glucosidase was performed. Most of these derivatives showed increased activity compared to acarbose as the positive control. The analysis of the SAR indicated that meta-chlorine substitution, as well as polar group with potential hydrogen interactions at the R position, was beneficial to α-glucosidase inhibition. The most potent candidate in this series 9d (IC 50 = 3.2 ± 0.3 µM) was chosen for further biological evaluation. The enzyme kinetics assessments indicated that compound 9d inhibited α-glucosidase in a competitive inhibition manner (Ki = 3.2 µM). According to the docking study, compound 9d was well fitted in the active site of α-glucosidase through both hydrophobic and hydrogen interactions. Overall, it can be understood that the most potent derivative (9d) exhibited better inhibitory activity against a-glucosidase compared to lead compounds including A, to G reported compared to positive control reported in Fig. 1. In silico assessments confirmed the critical role of benzimidazole and aryl-acetamides to participate in interactions with the binding site of an enzyme.
Regarding that T2DM is public health concern nowadays, the inhibition of α-glucosidase is considered an efficient approach to target T2DM. It was shown that quinoline-based-benzo[d]imidazole bearing different acetamides constructed a new nucleus which provided a significant role for α-glucosidase inhibition. However, to better extract the SARs of this set of compounds, in the future project, heteroaryl or aliphatic substituents at the R position will be synthesized. Also, bioisosteric replacement of benzo[d]imidazole with other heteroaromatic rings will increase our insight into the design of more potent α-glucosidase inhibitors.

Experimental
Chemistry. All the reagents were purchased from commercial sources. 1 H and 13 C NMR spectra were determined by a Bruker FT-400 MHz spectrometer in DMSO-d 6 . All the chemical shifts were reported as (δ) values ppm. The MS spectra were recorded using an Agilent 7890A spectrometer at 70 eV. CHNOS analysis was performed using ECS4010 Costech Company. IR spectra were obtained with a Nicolet, FR -IR Magna 550. Meltingpoint were also recorded using Kofler hot-stage apparatus. (2) 31 . To N, N-dimethylformamide (70.0 mmol) in the roundbottomed flask, phosphorus oxychloride (120.0 mmol) was added dropwise and the reaction mixture was stirred for 1 h at 0-5 °C. To this flask, N-phenylacetamide (30.0 mmol) was added and stirred for an extra 30 min followed by refluxing for 5-4 h under N 2 atmosphere. After the reaction was completed (TLC monitoring), the mixture was poured into crushed ice under constant stirring. The precipitate obtained was vacuum filtered, washed with water, air-dried, and recrystallized from EtOAc to give the 2-chloroquinoline-3-carbaldehyde. 32 . The reaction was initiated by stirring the mixture of 2-chloroquinoline-3-carbaldehyde 2 (1 mmol) and sodium sulfide (1 mmol) for 2 h at room temperature in dry DMF (50 mL). Then, the reaction mixture was poured into crushed ice and made acidic with acetic acid. The product was filtered off, washed with water, and dried to give the desired 2-mercaptoquinoline-3-carbaldehyde that was further purified by recrystallization in ethanol.

Synthesis of 3-(1H-benzo[d]imidazol-2-yl)quinoline-2-thiol (5) 33 . 2-Mercaptoquinoline-3-carbaldehyde
(1 mmol) and o-phenylenediamine (1.2 mmol) were dissolved in 2 mL DMF. Under stirring at room temperature, 1 mmol of sodium metabisulfite is added and allowed to react at 120 °C for about 4 h. After completion of the reaction, the mixture was precipitated in ice water, filtered, and dried at room temperature. 34 . To a solution of aniline derivatives (1 mmol) in DMF (4 mL), chloroacetylchloride was added at 0 °C. The mixture was stirred at room temperature for 5 h and poured into water and then filtered to get the 8a-r. The obtained solids were then filtered, dried, and recrystallized from ethanol. www.nature.com/scientificreports/ 35 . A mixture of 3-(1H-benzo[d]imidazol-2-yl)quinoline-2-thiol (1 mmol) and potassium carbonate (1.5 mmol) in DMF were stirred at room temperature for 15-20 min. Afterward, N-chloroacetyl-aniline (1.2 mmol) was added to the above reaction mixture and stirred for an extra 4-5 h. After completion of the reaction, ice-cold water was added to the reaction mixture and stirred for 20 min. The obtained solid was filtered and washed with cold water several times. The acquired crude solid was purified by recrystallization from ethanol.  13 Table 2. Docking scores and interactions of compounds against the α-glucosidase (PDB ID: 5NN8).    13  www.nature.com/scientificreports/